Paper published in Nature Chemical Biology explores “guardian of the genome”

TAMPA, Fla. – A USF study has demonstrated that reducing the flexibility of an important protein, p53, has dramatic effects on a cell’s ability to repair DNA, which could ultimately lead to the onset of cancer.

The study was published Nov. 2, 2014, in the advanced online edition of Nature Chemical Biology (http://www.nature.com/doifinder/10.1038/nchembio.1668) by lead authors Dr. Gary Daughdrill, associate professor in the department of Cell Biology, Microbiology, and Molecular Biology and member of the Florida Center of Excellence for Drug Discovery and Innovation at the University of South Florida, Dr. Alexander Loewer at the Berlin Institute for Medical Systems Biology, and Dr. Philipp Selenko at the Leibniz Institute of Molecular Pharmacology.

“The cells in your body are constantly being assaulted by internal and external agents that can damage your DNA and ultimately result in the onset of cancer,” said Daughdrill. “A sophisticated process that involves many different proteins has evolved to maintain the integrity of your genome by repairing DNA damage.”

One of the proteins central to this process is the human tumor suppressor p53, the “guardian of the genome,” because it helps the cell decide when to repair DNA damage, which ultimately suppresses the formation of tumors and inhibits the onset of cancer. p53 is a very flexible protein containing long segments that are constantly changing their shape.

“Proteins are large organic molecules that assume different shapes to accomplish the different functions that keep cells alive and healthy,” said Daughdrill. “In general, proteins can be grouped into two broad structural classes; ordered proteins that are relatively rigid and assume a small number of shapes to accomplish their functions and disordered proteins that are very flexible and assume an almost incomprehensible number of shapes to accomplish their functions.

“Most proteins are comprised of both ordered and disordered regions, and the protein chosen for our study fits into this category,” said Daughdrill. “It is called the ‘guardian of the genome’ because it helps cells decide how to respond to stressful situations. The inherent flexibility of p53 will determine whether cells will try to repair the DNA or undergo programmed cell death, also called apoptosis.”

Daughdrill’s research group at USF designed mutations in the transcriptional activation domain of p53 (p53TAD) that reduced its flexibility by making it more helical, a common shape for proteins that resembles a spiral staircase.

They were able to determine whether p53TAD was more or less helical using nuclear magnetic resonance (NMR) spectroscopy. USF’s Protein NMR Core Facility, which Daughdrill helped to develop, was critical to the success of the project.

Loewer’s research group in Berlin was able to put the mutant forms of full-length p53 into living cells and test how they responded to DNA damage.

The authors found that making p53 more helical (i.e. less flexible) increased its ability to interact with another important protein called Mdm2, and that increasing the interaction between p53 and Mdm2 reduced the cells’ ability to temporarily stop growing and make repairs to the genome.

According to Daughdrill, “the work is a significant step forward in our understanding of protein structure and function because it shows that a protein like p53, which is known to assume many different shapes in the test tube, is also assuming these different shapes inside living cells. It also showed that the flexibility of p53 is essential for its proper function.”

“This effort was truly interdisciplinary,” said Daughdrill. “The collective work performed at our three institutions allowed us to make predictions about how certain amino acid mutations impacted the inherent flexibility and function of p53 and to combine multiple techniques to track the effect of these mutations from the NMR tube to the cellular environment.”

Daughdrill was involved last year in a similar interdisciplinary research project with Dr. Kristina Schmidt, associate professor in USF’s Department of Cell Biology, Microbiology, and Molecular Biology, which was published in Nucleic Acids Research.

“Interdisciplinary collaborations,” said Daughdrill, “especially ones that combine computational biology, biophysics, cellular biology, and molecular biology, are absolutely necessary for solving the biggest problems in human health, many of which are caused by proteins like p53 that are capable of assuming a large number of shapes.”

Research supported by the Deutsche Forschungsgemeinschaft (Emmy Noether grant PS1794/1-1 to P.S.), the Association pour la Recherche contre le Cancer (postdoctoral fellowship to F.-X.T.), the European Union FP7 (Marie Curie CIG to A.L.), the American Cancer Society (RSG-07-289-01-GMC to G.W.D.) and the National Science Foundation (MCB-0939014 to G.W.D.).

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